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A wearable potentiometric device is reported based on an innovative butterfly-like paper-based microfluidic system, allowing for continuous monitoring of pH and Na+ levels in sweat during physical activity. Specifically, the use of the butterfly-like configuration avoids evaporation phenomena and memory effects, enabling precise and timely biomarker determination in sweat. Two ad hoc modified screen-printed electrodes were embedded in the butterfly-like paper-based microfluidics, and the sensing device was further integrated with a portable and miniaturized potentiostat, leveraging Bluetooth technology for efficient data transmission. First, the paper-based microfluidic configuration was tested for optimal fluidic management to obtain optimized performance of the device. Subsequently, the two electrodes were individually tested to detect the two biomarkers, namely pH and Na+. The results demonstrated highly promising near-Nernstian (0.056 ± 0.002 V/dec) and super-Nernstian (- 0.080 ± 0.003 V/pH) responses, for Na+ and pH detection, respectively. Additionally, several important parameters such as storage stability, interferents, and memory effect by hysteresis study were also investigated. Finally, the butterfly-like paper-based microfluidic wearable device was tested for Na+ and pH monitoring during the physical activity of three volunteers engaged in different exercises, obtaining a good correlation between Na+ increase and dehydration phenomena. Furthermore, one volunteer was tested through a cardiopulmonary test, demonstrating a correlation between sodium Na+ increase and the energetic effort by the volunteer. Our wearable device highlights the high potential to enable early evaluation of dehydration and open up new opportunities in sports activity monitoring.

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In cancer metastasis, where mortality rates remain high despite advancements in medical treatments, understanding the molecular pathways and cellular dynamics underlying tumor spread is critical for devising more effective therapeutic strategies. Here, a folding paper system was proposed and developed to mimic native tumor microenvironment. This system, composed of 7 stacked layers of paper enclosed in a holder, allows for the culture of cancer cells under conditions mimicking those found in solid tumors, including limited oxygen and nutrients. Because of the migratory capabilities of cancer cells, the cells in the center layer could migrated to outer layers of the paper stack, enabling the differentiation of cells based on their migratory potential. Subsequent gene expression analysis, conducted through RT-PCR and RNA sequencing, revealed significant correlations between cancer cell migration distance and the expression of genes associated with hypoxia, metabolism, ATP production, and cellular process. Moreover, our study identified cells with aggressive phenotypic traits from the outer layers of the paper stack, highlighting the potential of this system for enabling the study of aggressive cancer cell characteristics. Validation of the folding paper system against clinical carcinoma tissue demonstrated its ability to faithfully mimic the native tumor microenvironment. Overall, our findings underscore the utility of the folding paper system as a valuable tool for investigating and identifying critical molecular pathways involved in cancer metastasis.

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Wax printing is the most widely used method for fabricating microfluidic paper-based analytical devices (µPADs), but it still suffers from disadvantages like discontinuation of wax printers and need for additional equipment for heating treatment. To address these issues, this work initially describes a new class of wax printing approach for high-precision, batch fabrication of µPADs using a household 3D printer. It only involves a one patterning step of printing polyethylene wax into rice paper body. Under optimized parameters, a fabrication resolution, namely the minimum hydrophilic channel width, down to ~189 ± 30 µm could be achieved. In addition, the analytical applicability of such polyethylene wax-patterned µPADs was demonstrated well with enhanced colorimetric detection of dopamine as a model analyte by combining metal-organic framework (MOF) based nanoenzymes (ZIF-67) with a smartphone (for portable quantitative readout). The developed nanosensor could linearly detect dopamine over a concentration range from 10 to 1000 µM, with a detection limit of ca. 2.75 µM (3σ). The recovery results for analyzing several real samples (i.e., pig feed, chicken feed, pork and human serum) were between 91.82 and 102.79%, further validating its good detection accuracy for potential practical applications in food safety and medical diagnosis.

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Efficient diagnosis of mycobacterial infections can effectively manage and prevent the transmission of infectious diseases. Unfortunately, existing diagnostic strategies are challenged by long assay times, high costs, and highly specialized expertise to distinguish between pulmonary tuberculosis (PTB) and nontuberculous mycobacterial pulmonary diseases (NTM-PDs). Herein, in this study, an optimized 3D paper-based analytical device (µPAD) is incorporated with a closed lateral flow (LF) strip into a loop-mediated isothermal amplification (LAMP) device (3D-µPAD-LF-LAMP) for rapid, low-cost, and visual detection of pathogenic mycobacteria. The platform's microfluidic feature enhanced the nucleic acid amplification, thereby reducing the costs and time as compared to boiling, easyMAG, and QIAGEN techniques. Moreover, the LF unit is specifically designed to minimize aerosol contamination for a user-friendly and visual readout. 3D-µPAD-LF-LAMP is optimized and assessed using standard strains, demonstrating a limit of detection (LOD) down to 10 fg reaction-1 . In a cohort of 815 patients, 3D-µPAD-LF-LAMP displays significantly better sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and diagnostic accuracy than conventional bacterial culture and Xpert techniques. Collectively, 3D-µPAD-LF-LAMP demonstrates enhanced accessibility, efficiency, and practicality for the diagnosis of multiple pathogenic mycobacteria, which can be applied across diverse clinical settings, thereby ultimately improving public health outcomes.

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BACKGROUND: Developing disposable paper-based devices has positively impacted analytical science, particularly in developing countries. Some benefits of those devices include their versatility, affordability, environmentally friendly, and the possibility of being integrated with portable electrochemical or colorimetric detectors. Paper-based analytical devices (PADs) comprising circular zones and microfluidic networks have been successfully employed in the analytical chemistry reign. However, the combination of the stencil-printing method and alternative binder has not been satisfactorily explored for fabricating colorimetric paper devices. RESULTS: We developed PADs exploring the stencil printing approach and glass varnish as the hydrophobic chemical agent. As a proof-of-concept, the colorimetric assay of salivary α-amylase (sAA) was performed in saliva samples. Through the scanning electron microscopy measurements, it was possible to indicate satisfactory definitions between native fibers and barrier, and that the measured values for the channel width revealed suitable fidelity (R2 = 0.99) with the nominal widths (ranging from 400 to 5000 µm). The proposed hydrophobic barrier exhibited excellent chemical resistance. The analytical applicability for detecting sAA revealed linear behavior in the range from 2 to 12 U mL-1 (R2 = 0.99), limit of detection of 0.75 U mL-1, reproducibility (RSD ≤2.4%), recovery experiments ranged from 89 to 108% and AGREE response (0.86). In addition, the colorimetric analysis of sAA in four different saliva samples demonstrated levels ranging from 202 to 2080 U mL-1, which enabled monitoring the absence and presence of periodontitis. SIGNIFICANCE: This report has presented the first use of a self-adhesive mask and glass varnish for creating circular zones and microfluidic architectures on paper without using thermic or UV curing treatments. Also, the proposed analytical methodology for detecting sAA exhibited suitable ecological impact considering the AGREE tool. We believe the proposed fabrication of paper devices emerges as a novel, simple, high-fidelity microfluidic channel and portable analytical approach for colorimetric sensing.

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Identification of correct blood types holds paramount importance in understanding the pathophysiological parameters of patients, therapeutic interventions, and blood transfusion. Considering the wide applications of blood typing, the requirement of centralized laboratory facilities is not well suited on many occasions. In this context, there has been a significant development of such blood typing devices on different microfluidic platforms. The advantages of these microfluidic devices offer easy, rapid test protocols, which could potentially be adapted in resource-limited settings and thereby can truly lead to the decentralization of testing facilities. The advantages of pump-free liquid transport (i.e., low power consumption) and biodegradability of paper substrates (e.g., reduction in medical wastes) make it a more preferred platform in comparison to other microfluidic devices. However, these devices are often coupled with some inherent challenges, which limit their potential to be used on a mass commercial scale. In this context, our Review offers a succinct summary of the recent development, especially to understand the importance of underlying facets for long-term sustainability. Our Review also delineates the role of integration with digital technologies to minimize errors in interpreting the readouts.

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Periosteum-derived progenitor cells (PDPCs) are highly promising cell sources that are indispensable in the bone healing process. Adipose-derived stem cells (ADSCs) are physiologically close to periosteum tissue and release multiple growth factors to promote the bone healing process. Co-culturing PDPCs and ADSCs can construct periosteum-bone tissue microenvironments for the study of cellular crosstalk and molecular signal in the bone healing process. In the current work, a paper-based osteogenesis-on-a-chip platform was successfully developed to provide an in vitro three-dimensional coculture model. The platform was a paper substrate sandwiched between PDPC-hydrogel and ADSC-hydrogel suspensions. Cell secretion could be transferred through the paper substrate from one side to another side. Growth factors including BMP2, TGF-ß, POSTN, Wnt proteins, PDGFA, and VEGFA were directly analyzed by a paper-based immunoassay. Cellular crosstalk was studied by protein expression on the paper substrate. Moreover, osteogenesis of PDPCs was investigated by examining the mRNA expressions of PDPCs after culture. Neutralizing and competitive assays were conducted to understand the correlation between growth factors secreted from ADSCs and the osteogenesis of PDPCs. In vitro periosteum-bone tissue microenvironment was established by the paper-based osteogenesis-on-a-chip platform. The proposed approach provides a promising assay of cellular crosstalk and molecular signal in 3D coculture microenvironment that may potentially lead to the development of effective bone regeneration therapy.

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Developing paper-based electrophoretic methods involve dealing with significant uncertainty levels when compared to their capillary counterparts. Critical information for developing these kinds of methods are the electrophoretic mobility of background electrolytes and samples. This work presents the design and characterization of a device for measuring the electrophoretic mobilities of dyes in porous media. The device was developed with the aim of validating a previously presented model and also proposing a protocol for the straightforward determination of electrophoretic mobilities in porous media when open-channel values are already known. Whatman #1 paper was used as a model substrate as far as it is the most common porous medium substrate for paper-based electrophoresis. The device was designed using a numerical simulation-assisted approach, utilizing OpenFOAM® and specific solvers for capillary transport and electromigration, namely porousMicroTransport and electroMicroTransport, respectively. The electrophoretic mobilities of five dyes were analyzed experimentally with the proposed device. To establish appropriate comparative values at different pHs, experiments in fused silica capillaries were also performed. An effective parameter model for describing the electrophoretic behavior of dyes in porous media, that is, the constriction factor, was found consistent with previous reports for the Whatman #1 paper. This consistency was found after considering (via direct measurements) the chromatographic effect of the medium over each dye. Consequently, the recorded values hold significant worth due to their potential for direct application in designing new experiments or devices in Whatman #1 paper. With the validation of the model through the experiments with the proposed device, those researchers interested on developing electrophoretic methods in porous substrates can make use of the open-channel electrophoretic mobilities reported in the literature, or in the well-known software databases, and correct them for the media of interest just by performing two simple characterization steps.

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Rapid and accurate diagnosis of cardiovascular diseases (CVDs) at the earliest stage is of paramount importance to improve the treatment outcomes and avoid irreversible damage to a patient's cardiovascular system. Microfluidic paper-based devices (µPADs) represent a promising platform for rapid CVD diagnosis at the point of care (POC). This paper presents an electrochemical µPAD (E-µPAD) with an all-in-one origami design for rapid and POC testing of cardiac protein markers in whole blood. Based on the label-free, electrochemical impedance spectroscopy (EIS) immunoassay, the E-µPAD integrates all essential components on a single chip, including three electrochemical cells, a plasma separation membrane, and a buffer absorption pad, enabling easy and streamlined operations for multiplexed detection of three cardiac protein markers [cardiac troponin I (cTnI), brain natriuretic peptide (BNP)-32, and D-Dimer] on a finger-prick whole blood sample within 46 min. Superior analytical performance is achieved through sensitive EIS measurement on carbon electrodes decorated with semiconductor zinc oxide nanowires (ZnO NWs). Using spiked human plasma samples, ultralow limits of detection (LODs) of E-µPAD are achieved at 4.6 pg/mL (190 fM) for cTnI, 1.2 pg/mL (40 fM) for BNP-32, and 146 pg/mL (730 fM) for D-Dimer. Real human blood samples spiked with purified proteins are also tested, and the device's analytical performance was proven to be comparable to commercial ELISA kits. The all-in-one E-µPAD will allow rapid and sensitive testing of cardiac protein markers through easy operations, which holds great potential for on-site screening of acute CVDs in nonlaboratory settings such as emergency rooms, doctor's offices, or patient homes.

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Over the past ten years, microfluidic paper-based analytical devices (micro-PADs) have attracted a lot of attention as a viable analytical platform. It is expanding as a result of advances in manufacturing processes and device integration. Conventional microfluidics approaches have some drawbacks, including high costs, lengthy evaluation times, complicated fabrication, and the necessity of experienced employees. Hence, it is extremely important to construct a detection system that is quick, affordable, portable, and efficient. Nowadays, micro-PADs are frequently employed, particularly in electrochemical analyses, to replicate the classic standard laboratory experiments on a miniature paper chip. It has benefits like rapid assessment, small sample consumption, quick reaction, accuracy, and multiplex function. The goal of this review is to examine modern paper microfluidics-based electrochemical sensing devices for the detection of macromolecules, small molecules, and cells in a variety of real samples. The design and fabrication of micro-PADs using conventional and the latest techniques have also been discussed in detail. Lastly, the limitations and potential of these analytical platforms are examined in order to shed light on future research.

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The development of low-cost, disposable electrochemical sensors is an essential step in moving traditionally inaccessible quantitative diagnostic assays toward the point of need. However, a major remaining limitation of current technologies is the reliance on standardized reference electrode materials. Integrating these reference electrodes considerably restricts the choice of the electrode substrate and drastically increases the fabrication costs. Herein, we demonstrate that adoption of two-electrode detection systems can circumvent these limitations and allow for the development of low-cost, paper-based devices. We showcase the power of this approach by developing a continuous flow assay for urinary creatinine enabled by an embedded graphenic two-electrode detector. The detection system not only simplifies sensor fabrication and readout hardware but also provides a robust sensing performance with high detection efficiencies. In addition to enabling high-throughput analysis of clinical urine samples, our two-electrode sensors provide unprecedented insights into the fundamental mechanism of the ferricyanide-mediated creatinine reaction. Finally, we developed a simplified circuitry to drive the detector. This forms the basis of a smart reader that guides the user through the measurement process. This study showcases the potential of affordable capillary-driven cartridges for clinical analysis within primary care settings.

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Microfluidic paper-based analytical devices (µPADs) are indispensable tools for disease diagnostics. The integration of electronic components into µPADs enables new device functionalities and facilitates the development of complex quantitative assays. Unfortunately, current electrode fabrication methods often hinder capillary flow, considerably restricting µPAD design architectures. Here, laser-induced graphenization is presented as an approach to fabricate porous electrodes embedded into cellulose paper. The resulting electrodes not only have high conductivity and electrochemical activity, but also retain wetting properties for capillary transport. Paper-based electrofluidics, including a lateral flow device for injection analysis of alkaline phosphatase in serum and a vertical flow device for quantitative detection of HPV16 with a CRISPR-based assay are demonstrated. It is expected that this platform will streamline the development of diagnostic devices that combine the operational simplicity of colorimetric lateral flow tests with the added benefits and possibilities offered by electronic signaling.

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Neglected tropical diseases (NTDs) affect tropical and subtropical countries and are caused by viruses, bacteria, protozoa, and helminths. These kinds of diseases spread quickly due to the tropical climate and limited access to clean water, sanitation, and health care, which make exposed people more vulnerable. NTDs are reported to be difficult and inefficient to diagnose. As mentioned, most NTDs occur in countries that are socially vulnerable, and the lack of resources and access to modern laboratories and equipment intensify the difficulty of diagnosis and treatment, leading to an increase in the mortality rate. Portable and low-cost microfluidic systems have been widely applied for clinical diagnosis, offering a promising alternative that can meet the needs for fast, affordable, and reliable diagnostic tests in developing countries. This review provides a critical overview of microfluidic devices that have been reported in the literature for the detection of the most common NTDs over the past 5 years.

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Microfluidics is very crucial in lab-on-a-chip systems for carrying out operations in a large-scale laboratory environment on a single chip. Microfluidic systems are miniaturized devices in which the fluid behavior and control can be manipulated on a small platform, with surface forces on the platform being greater than volumetric forces depending on the test method used. In recent years, paper-based microfluidic analytical devices (µPADs) have been developed to be used in point-of-care (POC) technologies. µPADs have numerous advantages, including ease of use, low cost, capillary action liquid transfer without the need for power, the ability to store reagents in active form in the fiber network, and the capability to perform multiple tests using various measurement techniques. These benefits are critical in the advancement of paper-based microfluidics in the fields of disease diagnosis, drug application, and environment and food safety. Cancer is one of the most critical diseases for early detection all around the world. Detecting cancer-specific biomarkers provides significant data for both early diagnosis and controlling the disease progression. µPADs for cancer biomarker detection hold great promise for improving cure rates, quality of life, and minimizing treatment costs. Although various types of bioanalytical platforms are available for the detection of cancer biomarkers, there are limited studies and critical reviews on paper-based microfluidic platforms in the literature. Hence, this article aims to draw attention to these gaps in the literature as well as the features that future platforms should have.

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This paper describes the design and construction of dual microfluidic paper-based analytical devices (dual-µPADs) as a lab-on-paper platform involving a "do-it-yourself" fabrication protocol. The device comprises a colorimetric and electrochemical module to obtain a dual-mode signal readout sensing strategy. A 3D pen polymeric resin was used to prepare graphite carbon-based electrodes and hydrophobic barriers on paper substrates. The proposed carbon-based ink was employed to manufacture electrodes on paper based on a stencil-printing approach, which were further characterized by electrochemical and morphological analyses. The analytical performance of the dual-µPADs was simultaneously evaluated for lactate, pH, nitrite, and salivary amylase (sAA) analysis. To demonstrate the proof-of-concept, saliva samples collected from both healthy individuals and those with periodontitis were successfully tested to demonstrate the feasibility of the proposed devices. Samples collected from individuals previously diagnosed with periodontitis showed high levels of nitrite and sAA (> 94 µmol L-1 and > 610 U mL-1) in comparison with healthy individuals (≤ 16 µmol L-1 and 545 U mL-1). Moreover, periodontitis saliva resulted in acid solution and almost null lactate levels. Notably, this protocol supplies a simple way to manufacture dual-µPADs, a versatile platform for sensitive detecting of biomarkers in saliva playing a crucial role towards the point-of-care diagnosis of periodontal disease.

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Nanofibrillated cellulose paper (nanopaper) has gained growing interest as one promising substrate material for paper-based microfluidics, thanks to its ultrasmooth surface, high optical transparency, uniform nanofiber matrix with nanoscale porosity, and tunable chemical properties. Recently, research on nanopaper-based microfluidics has quickly advanced; however, the current technique of patterning microchannels on nanopaper (i.e., 3D printing, spray coating, or manual cutting and sticking), that is fundamental for application development, still has some limitations, such as ease-of-contamination, and more importantly, only enabling millimeter-scale channels. This paper reports a facile process that leverages the simple operations of microembossing with the convenient plastic micro-molds, for the first time, patterning nanopaper microchannels downing to 200 µm, which is 4 times better than the existing methods and is time-saving (<45 mins). We also optimized the patterning parameters and provided one quick look-up table as the guideline for application developments. As proof-of-concept, we first demonstrated two fundamental microfluidic devices on nanopaper, the laminar-mixer and droplet generator, and two functional nanopaper-based analytical devices (NanoPADs) for glucose and Rhodamine B (RhB) sensing based on optical colorimetry and surface-enhanced Raman spectroscopy, respectively. The two NanoPADs showed outstanding performance with low limits of detection (2 mM for glucose and 19fM for RhB), which are 1.25× and 500× fold improvement compared to the previously reported values. This can be attributed to our newly developed highly accurate microchannel patterning process that enables high integration and fine-tunability of the NanoPADs along with the superior optical properties of nanopaper.

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Since the inception of the first electrochemical devices on paper substrates, many different reports of microfluidic paper-based electroanalytical devices (µPEDs), innovative hydrophobic barriers and electrode fabrication processes have allowed the incorporation of diverse materials, resulting in different applications and a boost in performance. These advancements have led to the creation of paper-based devices with comparable performance to many standard conventional devices, with the added benefits of pumpless fluidic transport, component separation and reagent storage that can be exploited to automate and handle sample preprocessing. Herein, we review µPEDs, summarize the characteristics and functionalities of µPEDs, such as separation, fluid flow control and storage, and outline the conventional and emerging fabrication and modification approaches for µPEDs. We also examine the recent application of µPEDs in biomedicine, the environment, and food and water safety, as well as some limitations and challenges that must be addressed.

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Wearable sweat sensors, a product of the development of flexible electronics and microfluidic technologies, can continuously and noninvasively monitor abundant biomarkers in human sweat; however, sweat interferences, such as sebum, can reduce sensor reliability and accuracy. Herein, for the first time, the influence of sebum on the potentiometric response of an all-solid-state pH sensor was studied, and the obtained experimental results show that sebum mixed in sweat can decrease the potential response of the sensor and the slope of its calibration curve. A paper-based sandwich-structured pH sensor that can filter the sebum mixed in sweat was proposed based on commonly used oil-control sheets. Moreover, the hydrophilic properties, microstructure, and microfluidic performance of the sensor were investigated. The detection performance of the paper-based sandwich-structured pH sensor was comprehensively evaluated in terms of calibration in the presence of sebum and potentiometric response upon the addition of sebum. Furthermore, the anti-interference ability of the sensor was evaluated using different analytes under various deformation conditions. On-body trials were conducted to verify the performance, and their results showed that the proposed sensor can filter over 90% of the sebum in sweat, significantly enhancing sensor reliability and accuracy. Additionally, microfluidic channels could be simply fabricated using a scissor and paper, obviating the need for complex micromachining processes, such as photolithography and laser engraving. Overall, this work illustrates the influence of sebum on the detection performance of traditional potentiometric wearable sensors and paves the way for their development for real-world applications.

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This study describes the development of electrochemical paper-based analytical devices (ePADs) using carbon-based paste combining silicone glue and graphite powder. The ePADs were manufactured using the screen-printing technique, which consisted of depositing the conductive ink on a screencast on the paper surface. In addition, an alternative electrical connector was designed and 3D-printed to make the detection method cheaper, portable and reproducible. The morphological, structural, and electrochemical properties of the conductive material developed were investigated through scanning electron microscopy (SEM), Raman spectroscopy, electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV) measurements. The ePADs combined with the alternative connector revealed high repeatability, reproducibility, and stable responses considering a well-known redox probe ([Fe(CN)6]4-/3-). In addition, the proposed ePAD provided a linear response for standard solutions of ascorbic acid (AA) in the concentration range between 0.1 and 2.0 mmol L-1. The achieved limit of detection was 4.0 µmol L-1. As proof of applicability, the ePADs were evaluated for AA analysis in synthetic biofluids (blood plasma and urine), vitamin C tablets, and food (gelatine and orange juice) samples. The analytical parameters of the proposed device were compared with other reports in the literature and exhibited similar or even superior performance, highlighting its feasibility for sensing applications.

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Conventional detectors are mostly made up of complicated structures that are hard to use. A paper-based microfluidic chip, however, combines the advantages of being small, efficient, easy to process, and environmentally friendly. The paper-based microfluidic chips for biomedical applications focus on efficiency, accuracy, integration, and innovation. Therefore, continuous progress is observed in the transition from single-channel detection to multi-channel detection and in the shift from qualitative detection to quantitative detection. These developments improved the efficiency and accuracy of single-cell substance detection. Paper-based microfluidic chips can provide insight into a variety of fields, including biomedicine and other related fields. This review looks at how paper-based microfluidic chips are prepared, analyzed, and used to help with both biomedical development and functional integration, ideally at the same time.